Bioengineering of the Marine Diatom Phaeodactylum tricornutum with Cannabis Genes Enables the Production of the Cannabinoid Precursor, Olivetolic Acid.

The increasing demand for novel natural compounds has prompted the exploration of innovative approaches in bioengineering. This study investigates the bioengineering potential of the marine diatom Phaeodactylum tricornutum through the introduction of cannabis genes, specifically, tetraketide synthase (TKS), and olivetolic acid cyclase (OAC), for the production of the cannabinoid precursor, olivetolic acid (OA). P. tricornutum is a promising biotechnological platform due to its fast growth rate, amenability to genetic manipulation, and ability to produce valuable compounds. Through genetic engineering techniques, we successfully integrated the cannabis genes TKS and OAC into the diatom. P. tricornutum transconjugants expressing these genes showed the production of the recombinant TKS and OAC enzymes, detected via Western blot analysis, and the production of cannabinoids precursor (OA) detected using the HPLC/UV spectrum when compared to the wild-type strain. Quantitative analysis revealed significant olivetolic acid accumulation (0.6-2.6 mg/L), demonstrating the successful integration and functionality of the heterologous genes. Furthermore, the introduction of TKS and OAC genes led to the synthesis of novel molecules, potentially expanding the repertoire of bioactive compounds accessible through diatom-based biotechnology. This study demonstrates the successful bioengineering of P. tricornutum with cannabis genes, enabling the production of OA as a precursor for cannabinoid production and the synthesis of novel molecules with potential pharmaceutical applications.

Unravelling Novel Phytochemicals and Anticholinesterase Activity in Irish Cladonia portentosa.

Acetylcholinesterase inhibitors remain the mainstay of symptomatic treatment for Alzheimer's disease. The natural world is rich in acetylcholinesterase inhibitory molecules, and research efforts to identify novel leads is ongoing. Cladonia portentosa, commonly known as reindeer lichen, is an abundant lichen species found in Irish Boglands. The methanol extract of Irish C. portentosa was identified as an acetylcholinesterase inhibitory lead using qualitative TLC-bioautography in a screening program. To identify the active components, the extract was deconvoluted using a successive extraction process with hexane, ethyl acetate and methanol to isolate the active fraction. The hexane extract demonstrated the highest inhibitory activity and was selected for further phytochemical investigations. Olivetolic acid, 4-O-methylolivetolcarboxylic acid, perlatolic acid and usnic acid were isolated and characterized using ESI-MS and two-dimensional NMR techniques. LC-MS analysis also determined the presence of the additional usnic acid derivatives, placodiolic and pseudoplacodiolic acids. Assays of the isolated components confirmed that the observed anticholinesterase activity of C. portentosa can be attributed to usnic acid (25% inhibition at 125 µM) and perlatolic acid (20% inhibition at 250 µM), which were both reported inhibitors. This is the first report of isolation of olivetolic and 4-O-methylolivetolcarboxylic acids and the identification of placodiolic and pseudoplacodiolic acids from C. portentosa.

Identification and Optimization of more Efficient Olivetolic Acid Synthases.

Introduction: Olivetolic acid (OLA) is a key intermediate in cannabidiol (CBD) synthesis, and cannabinoids are important neuroactive drugs. However, the catalytic activity of olivetolic acid synthase (OLS), the key enzyme involved in OLA biosynthesis, remains low and its catalytic mechanism is unclear. Materials and Methods: In this study, we conducted a scrupulous screening of the pivotal rate-limiting enzyme and analyzed its amino acid sites that are critical to enzyme activity as validated by experiments. Results: Through stringent enzyme screening, we pinpointed a highly active OLS sequence, OLS4. Then, we narrowed down three critical amino acid sites (I258, D198, E196) that significantly influence the OLS activity. Conclusions: Our findings laid the groundwork for the efficient biosynthesis of OLA, and thereby facilitate the biosynthesis of CBD.

Plant-Produced Viral Nanoparticles as a Functionalized Catalytic Support for Metabolic Engineering.

Substrate channeling could be very useful for plant metabolic engineering; hence, we propose that functionalized supramolecular self-assembly scaffolds can act as enzymatic hubs able to perform reactions in close contiguity. Virus nanoparticles (VNPs) offer an opportunity in this context, and we present a functionalization strategy to display different enzymes on the outer surface of three different VNPs produced in plants. Tomato bushy stunt virus (TBSV) and Potato virus X (PVX) plant viruses were functionalized by the genetic fusion of the E-coil peptide coding sequence to their respective coat proteins genes, while the enzyme lichenase was tagged with the K-coil peptide. Immobilized E-coil VNPs were able to interact in vitro with the plant-produced functionalized lichenase, and catalysis was demonstrated by employing a lichenase assay. To prove this concept in planta, the Hepatitis B core (HBc) virus-like particles (VLPs) were similarly functionalized by genetic fusion with the E-coil sequence, while acyl-activating enzyme 1, olivetolic acid synthase, and olivetolic acid cyclase enzymes were tagged with the K-coil. The transient co-expression of the K-coil-enzymes together with E-coil-VLPs allowed the establishment of the heterologous cannabinoid precursor biosynthetic pathway. Noteworthy, a significantly higher yield of olivetolic acid glucoside was achieved when the scaffold E-coil-VLPs were employed.

Novel insights into the antibacterial activities of cannabinoid biosynthetic intermediate, olivetolic acid, and its alkyl-chain derivatives.

Investigations of antibacterial activities revealed that the incorporation of longer alkyl chains to the C-6 position in resorcylic acid conferred antibacterial properties against Staphylococcus aureus and Bacillus subtilis. The resultant olivetolic acid (OA) derivatives with n-undecyl and n-tridecyl side-chains, even those lacking the hydrophobic geranyl moiety from their C-3 positions, exhibited strong antibacterial activities against B. subtilis at a MIC value of 2.5 µM. Furthermore, the study demonstrated that the n-heptyl alkyl-chain modification at C-6 of cannabigerolic acid (CBGA) effectively enhanced the activity against B. subtilis, demonstrating the importance of the alkyl side-chain in modulating the bioactivity. Overall, the findings in this study provided insight into further evaluations of the antibacterial activities, as well as other various biological activities of OA and CBGA derivatives, especially with optimized hydrophobicities at both the alkyl and prenyl side-chain positions of the core skeleton for the discovery of novel drug seeds.

Olivetolic acid, a cannabinoid precursor in Cannabis sativa, but not CBGA methyl ester exhibits a modest anticonvulsant effect in a mouse model of Dravet syndrome.

OBJECTIVE: Cannabigerolic acid (CBGA), a precursor cannabinoid in Cannabis sativa, has recently been found to have anticonvulsant properties in the Scn1a+/- mouse model of Dravet syndrome. Poor brain penetration and chemical instability of CBGA limits its potential as an anticonvulsant therapy. Here, we examined whether CBGA methyl ester, a more stable analogue of CBGA, might have superior pharmacokinetic and anticonvulsant properties. In addition, we examined whether olivetolic acid, the biosynthetic precursor to CBGA with a truncated (des-geranyl) form, might possess minimum structural requirements for anticonvulsant activity. We also examined whether olivetolic acid and CBGA methyl ester retain activity at the epilepsy-relevant drug targets of CBGA: G-protein-coupled receptor 55 (GPR55) and T-type calcium channels. METHODS: The brain and plasma pharmacokinetic profiles of CBGA methyl ester and olivetolic acid were examined following 10 mg/kg intraperitoneal (i.p.) administration in mice (n = 4). The anticonvulsant potential of each was examined in male and female Scn1a+/- mice (n = 17-19) against hyperthermia-induced seizures (10-100 mg/kg, i.p.). CBGA methyl ester and olivetolic acid were also screened in vitro against T-type calcium channels and GPR55 using intracellular calcium and ERK phosphorylation assays, respectively. RESULTS: CBGA methyl ester exhibited relatively limited brain penetration (13%), although somewhat superior to that of 2% for CBGA. No anticonvulsant effects were observed against thermally induced seizures in Scn1a+/- mice. Olivetolic acid also showed poor brain penetration (1%) but had a modest anticonvulsant effect in Scn1a+/- mice increasing the thermally induced seizure temperature threshold by approximately 0.4°C at a dose of 100 mg/kg. Neither CBGA methyl ester nor olivetolic acid displayed pharmacological activity at GPR55 or T-type calcium channels. CONCLUSIONS: Olivetolic acid displayed modest anticonvulsant activity against hyperthermia-induced seizures in the Scn1a+/- mouse model of Dravet syndrome despite poor brain penetration. The effect was, however, comparable to the known anticonvulsant cannabinoid cannabidiol in this model. Future studies could explore the anticonvulsant mechanism(s) of action of olivetolic acid and examine whether its anticonvulsant effect extends to other seizure types.

Structure, function, and engineering of plant polyketide synthases.

Type III polyketide synthases (PKSs) catalyze sequential condensations of acyl-CoA thioesters to generate a variety of polyketide scaffolds with remarkable structural diversity and significant biological activities. These enzymes share a similar thiolase fold and use a common catalytic triad for the polyketide chain elongation and cyclization reactions. Structural and biochemical analyses of type III PKSs revealed that the functional diversity of these highly homologous enzymes is attributable to subtle changes in their active site volumes and architectures. The accumulated knowledge of their detailed catalytic versatility and mechanisms provides a platform for enzyme engineering via structure-guided approaches for the generation of unnatural novel polyketides. In this chapter, the methods for identification, biochemical characterization, and structure-guided engineering of polyketide synthases will be described in detail, along with brief overviews of the structures and functions of these enzymes.

High-Titer Production of Olivetolic Acid and Analogs in Engineered Fungal Host Using a Nonplant Biosynthetic Pathway.

The microbial synthesis of cannabinoids and related molecules requires access to the intermediate olivetolic acid (OA). Whereas plant enzymes have been explored for E. coli and yeast biosynthesis, moderate yields and shunt product formation are major hurdles. Here, based on the chemical logic to form 2,4-dihydroxybenzoate-containing natural products, we discovered a set of fungal tandem polyketide synthases that can produce OA and the related octanoyl-primed derivative sphaerophorolcarboxylic acid in high titers using the model organism Aspergillus nidulans. This new set of enzymes will enable new synthetic biology strategies to access microbial cannabinoids.

Structure of the Cannabis sativa olivetol-producing enzyme reveals cyclization plasticity in type III polyketide synthases.

In the native pathway to therapeutic cannabinoid biosynthesis in Cannabis sativa, the three-step production of a key intermediate, olivetolic acid, is catalysed by the enzymes tetraketide synthase (TKS; linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (OAC; final C2 â†’ C7 aldol condensation). In the absence of OAC, a nonenzymatic C2 â†’ C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. TKS is a type III polyketide synthase, and the question arises why it is unable to form olivetolic acid directly, but instead forms this unwanted side product. We determined the TKS, CoA complex structure, and performed structurally guided mutagenesis studies to identify potential residues responsible for cyclization pathway discrimination in type III polyketide synthases. Prior studies suggested an 'aldol switch' is necessary to allow linear tetraketide intermediate release prior to cyclization, thereby enabling subsequent olivetolic acid production by OAC. However, our studies do not support the presence of a universal or predictable 'aldol switch' consensus sequence. Instead, we propose the mode of ordered active site water activation between type III polyketide synthases catalysing different cyclization mechanisms is subtle and homologue-specific. Our work indicates that subtle structural variations between homologous enzymes can have a major mechanistic impact on the catalytic outcome. This highlights the importance of embedding high-resolution structural analysis of multiple enzyme homologues with classical site-directed mutagenesis studies when investigating highly similar enzymes with different mechanistic pathway outcomes. ENZYMES: TKS, EC 2.3.1.206; OAC, EC 4.4.1.26; chalcone synthase, EC 2.3.1.74; stilbene synthase, EC 2.3.1.95; 2-PS, EC 2.3.1.-. ACCESSION NUMBERS: The atomic coordinates and structure factors for the crystal structure of TKS have been deposited in the Protein Data Bank with accession number 6GW3.

Synthetic Pathway for the Production of Olivetolic Acid in Escherichia coli.

Type III polyketide synthases (PKS IIIs) contribute to the synthesis of many economically important natural products, most of which are currently produced by direct extraction from plants or through chemical synthesis. Olivetolic acid (OLA) is a plant secondary metabolite sourced from PKS III catalysis, which along with its prenylated derivatives has various pharmacological activities. To demonstrate the potential for microbial cell factories to circumvent limitations of plant extraction or chemical synthesis for OLA, here we utilize a synthetic approach to engineer Escherichia coli for the production of OLA. In vitro characterization of polyketide synthase and cyclase enzymes, OLA synthase and OLA cyclase, respectively, validated their requirement as enzymatic components of the OLA pathway and confirmed the ability for these eukaryotic enzymes to be functionally expressed in E. coli. This served as a platform for the combinatorial expression of these enzymes with auxiliary enzymes aimed at increasing the supply of hexanoyl-CoA and malonyl-CoA as starting and extender units, respectively. Through combining OLA synthase and OLA cyclase expression with the required modules of a ß-oxidation reversal for hexanoyl-CoA generation, we demonstrate the in vivo synthesis of olivetolic acid from a single carbon source. The integration of additional auxiliary enzymes to increase hexanoyl-CoA and malonyl-CoA, along with evaluation of varying fermentation conditions enabled the synthesis of 80 mg/L OLA. This is the first report of OLA production in E. coli, adding a new example to the repertoire of valuable compounds synthesized in this industrial workhorse.

Crystal structure of olivetolic acid: a natural product from Cetrelia sanguinea (Schaer.).

The title compound, C12H16O4 (systematic name: 2,4-dihy-droxy-6-pentyl-benzoic acid) is a natural product isolated from C. sanguinea (Schaer.) and is reported to have various pharmacological activities. The mol-ecule is approximately planar (r.m.s. deviation for the non-H atoms = 0.096 Å) and features an intra-molecular O-H⋯O hydrogen bond. In the crystal, each olivetolic acid mol-ecule is connected to three neighbours via O-H⋯O hydrogen bonds, generating (10-1) sheets. This crystal is essentially isostructural with a related resorcinolic acid with a longer alkyl chain.

Structural basis for olivetolic acid formation by a polyketide cyclase from Cannabis sativa.

In polyketide biosynthesis, ring formation is one of the key diversification steps. Olivetolic acid cyclase (OAC) from Cannabis sativa, involved in cannabinoid biosynthesis, is the only known plant polyketide cyclase. In addition, it is the only functionally characterized plant α+ß barrel (DABB) protein that catalyzes the C2-C7 aldol cyclization of the linear pentyl tetra-ß-ketide CoA as the substrate, to generate olivetolic acid (OA). Herein, we solved the OAC apo and OAC-OA complex binary crystal structures at 1.32 and 1.70 Å resolutions, respectively. The crystal structures revealed that the enzyme indeed belongs to the DABB superfamily, as previously proposed, and possesses a unique active-site cavity containing the pentyl-binding hydrophobic pocket and the polyketide binding site, which have never been observed among the functionally and structurally characterized bacterial polyketide cyclases. Furthermore, site-directed mutagenesis studies indicated that Tyr72 and His78 function as acid/base catalysts at the catalytic center. Structural and/or functional studies of OAC suggested that the enzyme lacks thioesterase and aromatase activities. These observations demonstrated that OAC employs unique catalytic machinery utilizing acid/base catalytic chemistry for the formation of the precursor of OA. The structural and functional insights obtained in this work thus provide the foundation for analyses of the plant polyketide cyclases that will be discovered in the future. DATA DEPOSITION: Structural data reported in this paper are available in the Protein Data Bank under the accession numbers 5B08 for the OAC apo, 5B09 for the OAC-OA binary complex and 5B0A, 5B0B, 5B0C, 5B0D, 5B0E, 5B0F and 5B0G for the OAC His5Q, Ile7F, Tyr27F, Tyr27W, Val59M, Tyr72F and His78S mutant enzymes, respectively.

Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa.

Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2-C7 intramolecular aldol cyclization of linear pentyl tetra-ß-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+ß barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Šresolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure-function relationship and catalytic mechanism of OAC.